Ver 75 decreased in GSH in the nuclear vs. only a 50 decrease of GSH within the cortical region.Impact of Suppressed GSH Levels on the Lens: Elevated ROSThe GSSG/GSH ratio, an index of ROS production, was mildly 
and strongly increased in HET-LESGKO vs. HOMLEGSKO mice, respectively (Fig. 1E). One of the major protein modifications associated with oxidative stress during human lens aging is methionine (-)-Indolactam V site sulfoxide (MetSOX), which increases with age and is highly elevated in cataractous lenses, with over 50 of membrane bound protein methionine present in oxidized form [15,19]. LC/MS based methionine sulfoxide determination indicated that the HOM-LEGSKO lenses protein bound methionine oxidation was significantly increased compared to heterozygous and wild type control lenses, and it was also elevated with age as measured at 9 months compared to 3 months of age (Fig. 1F). GSH antioxidative cycle enzymes glutathione reductase (GR) and glutathione peroxidase (GPx) activity were mildly but significantly decreased (Fig. 1G F). Surprisingly, however, we did not find elevation of protein modification by BI-78D3 supplier glycation in 1081537 HOM-LEGSKO mice (Fig.2), such as carboxymethyl-lysine (CML), carboxyethyllysine (CEL), methylglyoxal hydroimidazolone-1 (MG-H1), and glyoxal hydroimidazolone-1 (G-H1), which are known to positively correlate with aging in the human lens [20,21]. An explanation for this finding is provided in the Discussion. As the 40 reduction of GSH in the cortical region was predicted to increase oxidative stress, we investigated ROS production in living lenses ex vivo. Freshly isolated 6 months old HOM-LEGSKO and age matched control lenses were stained vitally with dihydrorhodamine 123 23115181 (DHR), a reactive oxygen species marker, and co-stained DNA with Hoechst 33342 to mark lens cell nuclei. Fluorescence (green) of DHR indicated much stronger ROS also present at cortical region of HOM-LEGSKO lens vs. age matched control lens (Fig.3).Results Conditional Deletion of Gclc Impairs Lens GSH SynthesisIn order to specifically delete Gclc from the lens, we crossed the Gclcfl/fl mice with MRL10-Cre mice [17] that express Cre recombinase in lens epithelia and fibers to ultimately generate mice homozygous 
for the conditional allele and hemizygous for MLR10 transgene. These mice Gclc2/2/MRL-10+/2 are deficient for Gclc specifically in the lens and are, herein named homozygous lens GSH knockout mice (HOM-LEGSKO). Similarly, the Gclcfl/+/MRL-10+/2 mice were named heterozygous lens GSH knockout mice (HET-LEGSKO) and should exhibit reduced Gclc levels in the lens. No lens abnormalities have been reported for mice that are hemizygous or homozygous for the MRL10-cre transgene in the absence of floxed alleles [17], and therefore phenotypes manifested in LEGSKO mice were contributed by Gclc deficiency alone. The LEGSKO mice were continuously crossbred with Gclcfl/fl mice (C57BL/6) to convert the genomic background towards C57BL/6. All the data provided in this paper are based on B6/FVB mixed background at third generation bred mice. The same breeding pattern and age matched control mice were used as wild type controls (Gclcfl/fl). LEGSKO mice exhibited reduced expression of Gclc transcripts and protein. HOM-LEGSKO lenses exhibited nearly undetectable levels of Gclc mRNA by real-time PCR, and Gclc transcripts were reduced nearly 50 in HET-LEGSKO lenses compared to wild type lenses (Fig. 1A). The levels of Gclc mRNA and protein were indistinguishable between Gclcfl/fl len.Ver 75 decreased in GSH in the nuclear vs. only a 50 decrease of GSH within the cortical region.Impact of Suppressed GSH Levels on the Lens: Elevated ROSThe GSSG/GSH ratio, an index of ROS production, was mildly and strongly increased in HET-LESGKO vs. HOMLEGSKO mice, respectively (Fig. 1E). One of the major protein modifications associated with oxidative stress during human lens aging is methionine sulfoxide (MetSOX), which increases with age and is highly elevated in cataractous lenses, with over 50 of membrane bound protein methionine present in oxidized form [15,19]. LC/MS based methionine sulfoxide determination indicated that the HOM-LEGSKO lenses protein bound methionine oxidation was significantly increased compared to heterozygous and wild type control lenses, and it was also elevated with age as measured at 9 months compared to 3 months of age (Fig. 1F). GSH antioxidative cycle enzymes glutathione reductase (GR) and glutathione peroxidase (GPx) activity were mildly but significantly decreased (Fig. 1G F). Surprisingly, however, we did not find elevation of protein modification by glycation in 1081537 HOM-LEGSKO mice (Fig.2), such as carboxymethyl-lysine (CML), carboxyethyllysine (CEL), methylglyoxal hydroimidazolone-1 (MG-H1), and glyoxal hydroimidazolone-1 (G-H1), which are known to positively correlate with aging in the human lens [20,21]. An explanation for this finding is provided in the Discussion. As the 40 reduction of GSH in the cortical region was predicted to increase oxidative stress, we investigated ROS production in living lenses ex vivo. Freshly isolated 6 months old HOM-LEGSKO and age matched control lenses were stained vitally with dihydrorhodamine 123 23115181 (DHR), a reactive oxygen species marker, and co-stained DNA with Hoechst 33342 to mark lens cell nuclei. Fluorescence (green) of DHR indicated much stronger ROS also present at cortical region of HOM-LEGSKO lens vs. age matched control lens (Fig.3).Results Conditional Deletion of Gclc Impairs Lens GSH SynthesisIn order to specifically delete Gclc from the lens, we crossed the Gclcfl/fl mice with MRL10-Cre mice [17] that express Cre recombinase in lens epithelia and fibers to ultimately generate mice homozygous for the conditional allele and hemizygous for MLR10 transgene. These mice Gclc2/2/MRL-10+/2 are deficient for Gclc specifically in the lens and are, herein named homozygous lens GSH knockout mice (HOM-LEGSKO). Similarly, the Gclcfl/+/MRL-10+/2 mice were named heterozygous lens GSH knockout mice (HET-LEGSKO) and should exhibit reduced Gclc levels in the lens. No lens abnormalities have been reported for mice that are hemizygous or homozygous for the MRL10-cre transgene in the absence of floxed alleles [17], and therefore phenotypes manifested in LEGSKO mice were contributed by Gclc deficiency alone. The LEGSKO mice were continuously crossbred with Gclcfl/fl mice (C57BL/6) to convert the genomic background towards C57BL/6. All the data provided in this paper are based on B6/FVB mixed background at third generation bred mice. The same breeding pattern and age matched control mice were used as wild type controls (Gclcfl/fl). LEGSKO mice exhibited reduced expression of Gclc transcripts and protein. HOM-LEGSKO lenses exhibited nearly undetectable levels of Gclc mRNA by real-time PCR, and Gclc transcripts were reduced nearly 50 in HET-LEGSKO lenses compared to wild type lenses (Fig. 1A). The levels of Gclc mRNA and protein were indistinguishable between Gclcfl/fl len.
